Orexin-1受体抑制剂对大鼠骨髓间充质干细胞增殖和成骨分化的影响

摘要

Objective To observe the effects of orexin-1 receptor inhibitor on proliferation and estrogenic differentiation of bone marrow mesenchymal stem cells ( BMSCs) . Methods BMSCs were isolated and cultured from Sprague-Dawley rats, and orexin-1 receptor inhibitor was prepared to different concentration of solutions:10-2 , 10-3 , 10-4 , 10-5 and 10-6 mol·L-1 . Then, the cells at passage 3 were cultured in orexin-1 receptor inhibitor solutions. The cell growth, alkaline phosphatase activity and osteocalcin secretion were measured by MTT, PNPP and ELISA, respectively. The mRNA expression levels of Runx2 and COL1A1 were detected by real-time quantitative PCR. Results The best concentration of orexin-1 receptor inhibitor for the proliferation of BMSCs was 1 × 10-4 mol·L-1 . Orexin-1 receptor inhibitor promoted the proliferation at concentration of 10-6 , 10-5 , 10-4 mol·L-1 in 3, 5 and 7 days. Orexin-1 receptor inhibitor at concentration of 10-6 , 10-5 and 10-4 mol·L-1 for 5 days significantly stimulated ALP activity. Orexin-1 receptor inhibitor significantly up-regulated Runx2 and COL1A1 mRNA expression at concentration of 10-6, 10-5, 10-4 mol·L-1 for 5 days (P<0.05). Conclusion Orexin-1 receptor inhibitor can promote BMSCs proliferation and stimulated ALP activity and osteocalcin secretion.%目的 探讨orexin-1受体抑制剂对骨髓间充质干细胞(BMSCs)增殖与成骨分化的影响.方法 提取SD大鼠BMSCs并鉴定,配制10-2,10-3,10-4,10-5,10-6 mol?L-1不同浓度的orexin-1受体抑制剂溶液,分别与第3代BMSCs共培养后,应用噻唑蓝(MTT)法检测orexin-1受体抑制剂对BMSCs增殖的影响.对硝基苯磷酸盐法和双抗体两步夹心酶联免疫吸附法检测碱性磷酸酶和骨钙素,实时荧光定量PCR法检测成骨分化相关基因Runx2、Col1a1 mRNA.结果orexin-1受体抑制剂最佳促BMSCs增殖浓度为1×10-4 mol?L-1.10-4,10-5,10-6 mol?L-1orexin-1受体抑制剂作用3,5,7 d能升高BMSCs胞浆内碱性磷酸酶表达,差异有统计学意义(P<0.05),而且这种作用在第5天达到高峰.10-4,10-5,10-6 mol?L-1 orexin-1受体抑制剂作用7 d可使骨钙素含量升高(P<0.05).10-4,10-5,10-6 mol?L-1 orexin-1受体抑制剂上调Runx2、COL1A1 mRNA表达.结论 orexin-1受体抑制剂可促进BMSCs增殖,同时促进其向成骨细胞方向分化,促进BMSCs表达碱性磷酸酶刺激骨钙素的分泌.